adopted on March 2007

Transcription

1 Statement of the Scientific Panel on Genetically Modified Organisms on the safe use of the nptii antibiotic resistance marker gene in genetically modified plants adopted on March BACKGROUND The Commission is currently considering the authorisation for placing on the market of the genetically modified (GM) potato line EH under Regulation (EC) No 1829/2003 on GM food and feed and under Directive 2001/18/EC, part C. The GM potato, developed for amylopectin production, also contains a nptii gene used as a selectable marker. The nptii gene codes for an aminoglycoside phosphotransferase conferring resistance to antibiotics such as kanamycin, neomycin, paromomycin, butirosin, gentamycin B and geneticin. Directive 2001/18/EC (EC, 2001) states that Member States and the Commission shall ensure that GMOs which contain genes expressing resistance to antibiotics in use for medical or veterinary treatment are taken into particular consideration when carrying out an environmental risk assessment. This is with a view to identify and phase out antibiotic resistance marker genes (ARMGs) in GMOs which may have adverse effects on human health and the environment. Over the last years, the use of antibiotic resistance marker genes for selection of GM plants, and which have been subject of safety assessment under Part C of the Directive 2001/18/EC, has been limited to the nptii gene. Some applications of GM plants submitted under Regulation No 1829/2003 also include the use of the nptii gene as a selectable marker, while in other cases the nptii gene has been excised after selection, or other markers such as the epsps and pat genes coding for herbicide tolerance were used. No other antibiotic resistance marker genes are currently present in applications submitted for approval. According to an earlier conclusion of the Scientific Panel on Genetically Modified Organisms (GMO Panel) (EFSA, 2004) 1 the use of nptii as a selectable marker in genetically modified plants and, more specifically, in the potato line EH (EFSA, 2006), does not pose a risk to the environment or to human and animal health. This conclusion was based on the low probability of gene transfer from plants to bacteria, the already widespread presence of the nptii gene in bacterial populations, and the limited use of kanamycin and neomycin in human and veterinary medicine. The Commission sought confirmation from the European Medicines Agency (EMEA) as to whether, notwithstanding a WHO working group classification of aminoglycosides as critically important antibacterials (WHO, 2005), the current or possible future uses of the antibiotics for which the nptii gene 1http:// 1

2 confers resistance is still in line with the earlier EFSA opinion. The EMEA was asked to consider whether the current or possible future medicinal uses of these antibiotics might have an impact on the earlier conclusions of the GMO Panel. In response to the Commission s request, the EMEA indicated that aminoglycosides comprise a class of antibiotics that has become increasingly important in the prevention and treatment of serious invasive bacterial infections in humans, since Gram-negative bacteria (and tuberculosis bacteria) are becoming resistant to other classes of antibiotics. The EMEA also stressed that, although kanamycin and neomycin are used relatively infrequently, the potential development of new chemical entities similar to kanamycin and neomycin should also be taken into account. In addition, although the veterinary use of kanamycin and neomycin is currently limited, aminoglycosides as a group are a class of antibiotics critically important for veterinary medicine. The EMEA considered that its competence did not extend to a detailed consideration of the likelihood of gene transfer of antibiotic resistance genes from plant material to bacteria of man and animals. Subsequently, the Commission requested EFSA (letter dated March 2, 2007) to consider the information provided by the EMEA and to indicate the potential consequences of the EMEA s conclusions on the safety assessment of the nptii gene and, where applicable, on the specific assessments of GMOs and derived food and feed. 2. ASSESSMENT A concern with respect to the presence of antibiotic resistance marker genes in GM plants is the potential for increased resistance to antibiotics in humans, animals and in organisms in the wider environment as a result of horizontal gene transfer. The safety assessment of the GMO Panel concerning the presence of the nptii gene in GM plants builds on a number of considerations. Key elements are the very low likelihood of transfer of a functional nptii gene (or any other gene), from GM plant material to microorganisms, and the prevalence of the nptii gene in bacterial clinical isolates and in the environment Likelihood of transfer of the nptii gene from the genome of GM plants to bacteria In considering the probability of functional gene transfer from plants into bacteria in the environment or human/animal gut, several aspects need to be taken into account: (i) (ii) (iii) DNA is released from plant material by normal digestion processes that take place in the gastrointestinal tract, or by activities of nucleases present in various organisms in the environment. The probability that bacteria will be exposed to DNA stretches long enough to contain the intact nptii gene is very low because of the above mentioned digestion and degradation processes (Lorenz and Wackernagel, 1994). The nptii gene from plant material can only be taken up by competent bacteria via natural transformation, a process that occurs infrequently in many bacteria and in most environmental conditions (Davison, 1999). 2

3 (iv) (v) (vi) If the intact nptii gene enters the bacteria, it will be rapidly degraded by restriction endonucleases in many bacterial cells which possess DNA restriction systems in order to destroy foreign DNA (Davison, 1999). If the intact nptii gene does indeed survive, the probability of its incorporation into the bacterial genome is very low unless there are homologous regions already present in the bacterial genome. Gene transfer from plants to bacteria has only been demonstrated under laboratory conditions when regions of homology were already present in the recipient bacterium (Bennett et al., 2004, de Vries et al., 2001, de Vries and Wackernagel, 2002, Kay et al., 2002, Tepfer et al., 2003). Expression of the incorporated nptii gene is unlikely considering that in GM plant material the nptii gene is under the control of a promoter with preferential expression in plants, which does not support its efficient expression in bacteria. (vii) Stable integration and inheritance of the nptii gene in the host bacterium is not likely in the absence of selective pressure from a relevant antibiotic. When all of the above mentioned aspects are taken into account, the probability of functional gene transfer from plants into microorganisms is extremely low. It is not surprising that transfer of an antibiotic resistance marker from GM plants to bacteria has not been observed under natural conditions (Gay and Gillespie, 2005). The EMEA has indicated that under laboratory conditions gene transfer from plants to bacteria has been demonstrated. EFSA has addressed this issue more extensively in its Opinion of 2004 (section 4) (EFSA, 2004). Gene transfer from plants to bacteria has only been demonstrated in a few highly transformable bacterial species (e.g., Acinetobacter sp. BD413 or Pseudomonas stutzeri) under artificial and forced laboratory conditions when regions of homology were already present in the recipient bacterium (Bennett et al., 2004, de Vries et al., 2001, de Vries and Wackernagel, 2002, Kay et al., 2002, Tepfer et al., 2003). In the absence of this optimisation of the process and selection pressure, resistance gene transfer from GM plants to bacteria, even in the laboratory, could not be demonstrated (Gebhard and Smalla, 1998) Prevalence of the nptii gene in soil, humans and animals As indicated in the Opinion (of the GMO Panel) on the use of antibiotic resistance genes as markers in GM plants, antibiotic resistance is a common feature in natural microbial communities in soils, aquatic systems, and habitats associated with animals and humans (EFSA, 2004). There is already a widespread presence of nptii in the soil environment as evidenced from DNA-based work with nptii as a probe in different locations in Western Europe (NSCFFS, 2005, Smalla et al., 1993) and in the USA (Leff et al., 1993). Studies indicate that, as expected of a gene located on a transposable genetic element, nptii is located on a wide range of replicons in bacterial clinical isolates from humans (Alvarez and Mendoza, 1992, Chang et al., 1992, Flamm et al., 1993). The nptii gene was present in 2.5% of bacterial clinical isolates resistant to kanamycin and neomycin collected between 1987 and 1991 in several European and Central and South American countries (Shaw et al., 1993). Studies on the prevalence of the nptii gene in animal-associated bacterial populations have not been found in the scientific literature. 3

4 2.3. Contribution of the nptii gene to the prevalence of resistance to kanamycin Kanamycin-resistant bacteria are ubiquitous in nature. Selective plating of different environmental samples on kanamycin-containing medium reduced the microbial count from 10 7 to 10 4 CFU/g (Smalla and van Elsas, 1996, Smalla et al., 1993). Only a fraction of kanamycin-resistant bacteria contain the nptii (aph(3 )- IIa) gene, the other resistant bacteria having different genes and/or other mechanisms conferring kanamycin resistance. The nptii gene has been reported to occur naturally only in eubacteria. In one survey, 3 out of 184 kanamycin resistant bacterial isolates from three stream sites in the USA (Leff et al., 1993) and 44 out of 355 from different habitats in the Netherlands (Smalla et al., 1993) contained nptii sequences Potential mutations of the nptii gene resulting in resistance to other antibiotics As reported in the opinion of the GMO Panel on antibiotic resistance genes as markers in GM plants (EFSA, 2004), resistance towards amikacin, an important reserve antibiotic could be obtained under laboratory conditions and was the result of a mutated nptii gene and a diminished rate of amikacin uptake into the bacterial cell (Perlin and Lerner, 1986). The increased affinity of a mutated nptii gene product for amikacin was later confirmed by site-directed mutagenesis which resulted in one altered nucleotide in the gene and an eight-fold increase in amikacin resistance in E.coli (Kocabiyik and Perlin, 1992). It has been suggested that the increased affinity for amikacin conferred by this mutation, might impair the clinical effectiveness of the drug. However, to date no clinical amikacin resistant strains with a mutated nptii gene have been identified. 3. CONCLUSIONS The GMO Panel agrees with the EMEA that the preservation of the therapeutic potential of the aminoglycoside group of antibiotics is important. The Panel is also of the opinion that the therapeutic effect of these antibiotics will not be compromised by the presence of the nptii gene in GM plants, given the extremely low probability of gene transfer from plants to bacteria and its subsequent expression. Furthermore, the GMO Panel considers it very unlikely that the presence of the nptii gene in GM plants will change the existing widespread prevalence of this antibiotic resistance gene in bacterial sources in the environment. The GMO Panel also points to evidence which indicates that integration of the nptii gene would only be one of many mechanisms by which bacteria could become resistant to aminoglycosides such as kanamycin. Therefore, the GMO Panel reiterates its earlier conclusions (EFSA, 2004) that the use of the nptii gene as selectable marker in GM plants (and derived food or feed) does not pose a risk to human or animal health or to the environment. The GMO Panel also confirms earlier safety assessments of GM plants and derived food/feed comprising the nptii gene. The GMO Panel emphasizes that the use of antibiotic resistance marker genes in GM plants has been the subject of several reviews (Gay and Gillespie, 2005, Goldstein et al., 2005, Miki and McHugh, 2004, Nap et al., 1992, Nielsen et al., 1998, Ramessar et al., 2007) and expert consultations: Working Party of the British Society for Antimicrobial Chemotherapy (Bennett et al., 2004), FAO/WHO Consultation on Foods Derived from Biotechnology (FAO/WHO, 2000), Scientific Steering Committee of the European Commission (SSC, 1999) Zentrale Kommission für die Biologische Sicherheit, DE (ZKBS, 1999), The Advisory Committee on 4

5 Novel Foods and Processes, UK (ACNFP, 1996). It has been concluded in these reports that the frequencies of gene transfer from plants to bacteria are likely to be extremely low and that the presence of antibiotic resistance marker genes, and in particular the nptii gene, in GM plants do not pose a relevant risk to human or animal health or to the environment. DOCUMENTATION PROVIDED TO EFSA 1. Letter from DG SANCO, dated 2 March 2007, concerning the presence of the nptii resistance gene in genetically modified organisms (ref. SANCO/E1/SG/cc (2007)D/510137). 2. Document from the EMEA Committee for Medicinal Products for Veterinary Use and Committee for Medicinal Products for Human Use, dated 22 February 2007, entitled Presence of the antibiotic resistance marker gene nptii in GM plants for food and feed uses, (ref. EMEA/CVMP/56937/2007-Final). REFERENCES ACNFP (1996) The use of antibiotic resistance markers in genetically modified plants for human food. The Advisory Committee on Novel Foods and Processes, UK. ALVAREZ, M. & MENDOZA, M. C. (1992) Epidemiological survey of genes encoding aminoglycoside phosphotransferases APH (3') I and APH (3') II using DNA probes. J. Chemother., 4, BENNETT, P. M., LIVESEY, C. T., NATHWANI, D., REEVES, D. S., SAUNDERS, J. R. & WISE, R. (2004) An assessment of the risks associated with the use of antibiotic resistance genes in genetically modified plants: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J. Antimicrob. Chemother., 53, CHANG, S. F., CHANG, L. L., CHOW, T. Y., WU, W. J. & CHANG, J. C. (1992) Prevalence of transposons encoding kanamycin, ampicillin and trimethoprim resistance in isolates from urinary tract infections detected using DNA probes. Gaoxiong Yi Xue Ke Xue Za Zhi, 8, DAVISON, J. (1999) Genetic exchange between bacteria in the environment. Plasmid, 42, DE VRIES, J., MEIER, P. & WACKERNAGEL, W. (2001) The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells. FEMS Microbiol. Lett., 195, DE VRIES, J. & WACKERNAGEL, W. (2002) Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination. Proc. Natl. Acad. Sci. USA, 99, EFSA (2004) Opinion of the Scientific Panel on Genetically Modified Organisms on the use of antibiotic resistance genes as marker genes in genetically modified plants (Question N EFSA-Q ). The EFSA Journal, 48,

6 EFSA (2006) Opinion of the Scientific Panel on Genetically Modified Organisms on an application (Reference EFSA-GMO-UK ) for the placing on the market of genetically modified potato EH with altered starch composition, for production of starch and food/feed uses, under Regulation (EC) No 1829/2003 from BASF Plant Science. The EFSA Journal, 324, FAO/WHO (2000) Safety aspects of genetically modified foods of plant origin. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology., World Health Organization. FLAMM, R. K., PHILLIPS, K. L., TENOVER, F. C. & PLORDE, J. J. (1993) A survey of clinical isolates of Enterobacteriaceae using a series of DNA probes for aminoglycoside resistance genes. Mol. Cell. Probes, 7, GAY, P. B. & GILLESPIE, S. H. (2005) Antibiotic resistance markers in genetically modified plants: a risk to human health? Lancet Infect. Dis., 5, GEBHARD, F. & SMALLA, K. (1998) Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol., 64, GOLDSTEIN, D. A., TINLAND, B., GILBERTSON, L. A., STAUB, J. M., BANNON, G. A., GOODMAN, R. E., MCCOY, R. L. & SILVANOVICH, A. (2005) Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies. J. Appl. Microbiol., 99, KAY, E., VOGEL, T. M., BERTOLLA, F., NALIN, R. & SIMONET, P. (2002) In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria. Appl. Environ. Microbiol., 68, KOCABIYIK, S. & PERLIN, M. H. (1992) Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3'-phosphotransferase-II. FEMS Microbiol. Lett., 72, LEFF, L. G., DANA, J. R., MCARTHUR, J. V. & SHIMKETS, L. J. (1993) Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA. Appl. Environ. Microbiol., 59, LORENZ, M. G. & WACKERNAGEL, W. (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev., 58, MIKI, B. & MCHUGH, S. (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotechnol., 107, NAP, J. P., BIJVOET, J. & STIEKEMA, W. J. (1992) Biosafety of kanamycin-resistant transgenic plants. Transgenic Res., 1, NIELSEN, K. M., BONES, A. M., SMALLA, K. & VAN ELSAS, J. D. (1998) Horizontal gene transfer from transgenic plants to terrestrial bacteria--a rare event? FEMS Microbiol. Rev., 22, NSCFFS (2005) An assessment on potential long-term health effects caused by antibiotic resistance marker genes in genetically modified organisms based on antibiotic usage and resistance patterns in Norway. Report from an Ad Hoc Group appointed by the Norwegian Scientific Panel on Genetically Modified Organisms and Panel on Biological Hazards. Norwegian Scientific Committee for Food Safety. 6

7 PERLIN, M. H. & LERNER, S. A. (1986) High-level amikacin resistance in Escherichia coli due to phosphorylation and impaired aminoglycoside uptake. Antimicrob. Agents Chemother., 29, RAMESSAR, K., PEREMARTI, A., GOMEZ GALERA, S., NAQVI, S., MORALEJO, M., MUÑOZ, P., CAPELL, T. & CHRISTOU, P. (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants. A case of the science not supporting the politics. Transgenic Res. (in press). SHAW, K. J., RATHER, P. N., HARE, R. S. & MILLER, G. H. (1993) Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev., 57, SMALLA, K. & VAN ELSAS, J. D. (1996) Monitoring genetically modified organisms and their recombinant DNA in soil environments. IN TOMIUK, J., WÖHRMANN, K. & SENTKER, A. (Eds.) Transgenic organisms - biological and social implications. Basel, Switzerland, Birkhäuser Verlag. SMALLA, K., VAN OVERBEEK, L. S., PUKALL, R. & VAN ELSAS, J. D. (1993) Prevalence of nptii and Tn5 in kanamycin-resistant bacteria from different environments. FEMS Microbiol. Ecol., 13, SSC (1999) Opinion of the Scientific Steering Committee on Antimicrobial Resistance. European Commission. Directorate-General XXIV. TEPFER, D., GARCIA-GONZALES, R., MANSOURI, H., SERUGA, M., MESSAGE, B., LEACH, F. & PERICA, M. C. (2003) Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer. Transgenic Res., 12, WHO (2005) Critically Important Antibacterial Agents for Human Medicine for Risk Management Strategies of Non-Human Use. Report of a WHO working group consultation, February 2005, Canberra, Australia., World Health Organization. ZKBS (1999) Stellungnahme der ZKBS zur Biologischen Sicherheit von Antibiotika-Resistenzgenen im Genom gentechnisch veränderter Pflanzen. Zentrale Kommission für die Biologische Sicherheit, DE, Gentechnik/093 ZKBS/01 Allg Stellun gnahmen/01 generellethemen/generellethemen node.html nnn=true. SCIENTIFIC PANEL MEMBERS Hans Christer Andersson, Salvatore Arpaia, Detlef Bartsch, Josep Casacuberta, Howard Davies, Lieve Herman, Marc De Loose, Niels Hendriksen, Sirpa Kärenlampi, Jozsef Kiss, Ilona Kryspin-Sørensen, Harry Kuiper, Ingolf Nes, Nickolas Panopoulos, Joe Perry, Annette Pöting, Joachim Schiemann, Willem Seinen, Jeremy Sweet, and Jean-Michel Wal. ACKNOWLEDGEMENT The GMO Panel wishes to thank Hans Jörg Buhk, Patrick Du Jardin, Boet Glandorf, John Heritage, Fergal O Gara, Armine Sefton, Philippe Vain, and Jan Dick van Elsas for their contributions to the statement. 7